Project Summary Streptococcus sanguinis is an oral bacterium that is normally considered a contributor to good oral health, but it has also been implicated as a major etiological agent of infective endocarditis (IE). IE is a serious disease caused by certain bacteria entering the bloodstream through lesions in the oral mucosa during surgery, mastication, or routine hygiene practices. Bacteria that are capable of binding to the heart valve can then proliferate, which may lead to congenital heart failure, aneurysm, or stroke. Currently, the only preventative for IE is the prescription of broad spectrum antibiotics given before dental procedures. With the increase in antibiotic resistance as well as the possibility of IE from routine hygiene activities, other preventatives are being investigated. ABC-family manganese transporters have been implicated as virulence factors for streptococcal IE and thus are putative drug targets. A secondary manganese transporter of S. sanguinis was recently identified and implicated as contributing to virulence in IE. This transporter is homologous to the ZIP family of proteins, which are traditionally associated with zinc and iron transport. This study seeks to determine the role of the secondary transporter through its regulation, as well as to investigate the amino acids responsible for manganese specificity. Aim 1 of the proposal will assess the regulation of the transporter under various oxygen, pH, and metal conditions. In Aim 2, the amino acids responsible for metal transport will be identified using site-directed mutagenesis to mutate specific residues of interest within S. sanguinis. Aim 3 will determine the metal specificity of the transporter by heterologous production and isolation of the protein, incorporation into liposomes, and assessment of its uptake specificity towards physiologically relevant transition metals by inductively coupled plasma mass spectrometry. Through these aims, the role and function of the transporter will be elucidated, enhancing our understanding of ZIP proteins and manganese-transporting proteins, especially as they relate to bacterial pathogenesis and oral competitiveness. Access and training for the appropriate instrumentation will be available for the approaches proposed in these aims in facilities within the Virginia Commonwealth University (VCU) Philips Institute for Oral Health Research, Department of Chemistry, and School of Pharmacy, as well as at Purdue University and Harvard University. Collaborators and mentors at VCU, Purdue University, and Harvard University will provide support and expertise in microbiology, bioinorganic chemistry, and molecular characterization of membrane proteins. Under the guidance of faculty members in each of these departments and institutions, this project will enhance the applicant?s knowledge of bioinorganic chemistry, protein characterization, and gene regulation as they relate to bacterial metal transport proteins and their contribution to pathogenesis and competitiveness.